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Background Lymphoblastic lymphoma (LBL) and acute lymphoblastic leukemia (ALL) are categorized as the same entity under precursor lymphoid neoplasms in the World Health Organization classification. However, compared to B-cell ALL, the molecular genetic makeup of B-cell LBL remains to be understood, mainly due to its rarity. We performed whole exome sequencing (WES) on seven patients with TCF3-PBX1-positive B-cell LBL. Methods WES was performed using DNA extracted from tumor specimens and paired blood samples at remission for six patients, and tumor-only analysis was performed for one patient whose remission sample was not available. For one patient, a relapsed sample was also analyzed. Results KMT2D variants and 6q LOH were found as recurrent alterations. Somatic variants of KMT2D were identified in three of the seven patients. Of note, the two patients with heterozygous nonsense variant of KMT2D were at stage III, without bone marrow infiltration. 6q LOH was also identified in two others, out of the seven patients. The common 6q deleted region of the two patients ranged from 6q12 to 6q16.3. Both patients had bone marrow infiltration. Analysis of recurrent case also revealed that the relapsed clone might be derived from a minor clone of the bone marrow at diagnosis. Conclusion In this study, through WES for seven patients with TCF3-PBX1-positive B-LBL, we identified KMT2D mutations and 6q LOH as recurrent alterations. In order to elucidate the relationship between these recurrent alterations and disease specificity or outcomes, further studies comparing with TCF3-PBX1-positive B-ALL are required.

Background/aim Tacrolimus is an essential immunosuppressant for successful allogeneic haematopoietic stem cell transplantation (Allo-HSCT). This study aimed to examine the change in the blood concentration of tacrolimus during switching from intravenous to oral administration in allo-HSCT for paediatric cancer to predict the optimal dosage. Patients and methods We retrospectively examined the medical records of 63 patients who received allo-HSCT and were administered tacrolimus. To compare bioavailability among different dose ranges, the blood concentration was divided by the dose (C/D). Results Thirty-nine patients (age range=children 1-15 years, adults 17-67 years) were switched to oral administration of tacrolimus. The C/D after switching was significantly lower in children than in adults (p=0.039). There was a strong positive correlation between age and C/D in children, whereas no correlation was observed in adults. Conclusion In paediatric cancer patients, switching tacrolimus administration route may result in reduced blood concentrations. This tendency is more prominent in younger children.

BACKGROUND Transient abnormal myelopoiesis (TAM) is a unique myeloproliferative disorder that occurs in neonates with constitutional trisomy 21/Down syndrome (DS). Although TAM also develops in neonates without constitutional trisomy 21, the clinical, cytogenetic, and molecular characteristics of those patients are not fully understood. PROCEDURE We retrospectively evaluated the clinical and cytogenetic findings and GATA1 mutation status of 17 neonates with TAM and nonconstitutional trisomy 21 tested for GATA1 mutations at our institute, and compared the findings with those of 64 neonates with TAM and constitutional trisomy 21/DS. RESULTS DS clinical features were observed in five of the 17 (29%) patients. In all patients, both trisomy 21 and GATA1 mutations were detected in diagnostic samples. Over a median follow-up of 33 (range, 0-139) months, early death (

Vitamin K deficiency in pregnant women causes intracranial hemorrhage (ICH) in fetuses. Fetal ICH frequently causes life-threatening and persistent neurological damage. However, indicators for preventing fetal ICH are not established. Two pregnant women developed long-term eating disorders caused by psychosis. They were administered intravenous fluid and vitamin supplementation, excluding vitamin K. The intracranial low-hypoechoic area on fetal ultrasound was suggestive of fetal ICH due to vitamin K deficiency. Their neonates showed severe developmental delay. Laboratory analysis revealed a normal prothrombin time, but elevated protein induced by vitamin K absence II. Pregnant women who have eating disorders more than 3 weeks could develop fetal ICH due to maternal subclinical vitamin K deficiency. Illness duration and protein induced by vitamin K absence II of pregnant woman may be indicators for vitamin K administration to prevent fetal intracranial hemorrhage.

Idiopathic pulmonary hemosiderosis (IPH) is a rare and life-threatening disorder. Early diagnosis and appropriate management are essential for their better prognosis and patients’ quality of life (QOL). It is considered that Down syndrome patients with IPH have a worse prognosis compared to other IPH cases. A 2-year-old girl with Down syndrome received the diagnosis of IPH after two episodes of massive pulmonary hemorrhage requiring assist ventilation, who suffered from recurrent IPH during tapering period of oral corticosteroid, started liposteroid therapy. We report here a case of successful control of recurrent IPH and improved QOL enormously with tapering dose of corticosteroid after starting liposteroid therapy.

The efficacy of and indications for hematopoietic stem cell transplantation (HSCT) in pediatric Langerhans cell histiocytosis (LCH) remain undetermined. This retrospective study analyzed 30 children with refractory LCH who underwent HSCT in Japan between 1996 and 2014. Eleven patients received a myeloablative conditioning (MAC) regimen, while 19 patients received a reduced-intensity conditioning (RIC) regimen. Among the 26 patients with complete data, 23 patients had risk organ (RO) involvement during clinical course. Disease status at HSCT was no active disease (NAD) (4), active disease-regression (AD-r) (2), active disease-stable (AD-s) (4), and active disease-progressive (AD-p) (16). Seventeen of the 30 patients (57%) were alive with a median follow-up of 433 days (range 9–5307) after HSCT. Death occurred within 3 months after HSCT in eight of 13 patients. RIC and MAC patients were similar in both overall survival (OS) (56.8% vs. 63.6%, respectively, p = 0.789) and failure-free survival (56.8% vs. 54.6%, respectively, p = 0.938). Regarding disease status at HSCT, the six patients with NAD/AD-r experienced better outcomes than the 20 with AD-s/AD-p (5-year OS, 100% vs. 54.5%, respectively, p = 0.040). Disease state at the time of HSCT was the most important prognostic factor.

Acute megakaryoblastic leukemia in children without Down syndrome (non-DS AMKL) is considered to be a poor prognostic subtype in acute myeloid leukemia. Recently, some chimeric fusion genes were found in pediatric non-DS AMKL; therefore, we attempted to detect chimeric fusion genes RBM15-MKL1, CBFA2T3-GLIS2, and NUP98-KDM5A from 10 pediatric non-DS AMKL diagnostic samples using polymerase chain reaction and Sanger sequencing methods. Two samples were positive for RBM15-MKL1, four had CBFA2T3-GLIS2, and only one case had NUP98-KDM5A. Both RBM15-MKL1-positive patients showed long-term remission after chemotherapy. The eight RBM15-MKL1-negative patients received hematopoietic stem cell transplantation (HSCT). In four CBFA2T3-GLIS2-positive patients, three had HSCT without complete remission and two of themdied. Additional treatment stratification depending on chimeric fusion genes and development of new therapeutic drugs are required for non-DS AMKL.

Unrelated cord blood transplantation in aplastic anemia: is anti-thymocyte globulin indispensable for conditioning?

This study reviewed the clinical characteristics of 112 pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients with TCF3-PBX1 fusion treated according to the Japan Association of Childhood Leukemia Study (JACLS) ALL02 protocol (n = 82) and Children's Cancer and Leukemia Study Group (CCLSG) ALL 2004 protocol (n = 30). The 3-year event-free survival (EFS) and overall survival (OS) rates were 85.4 ± 3.9% and 89.0 ± 3.5% in JACLS cohort, and the 5-year EFS and OS were 82.8 ± 7.0% and 86.3 ± 6.4% in CCLSG cohort, respectively, which are comparable to those reported in western countries. Conventional prognostic factors such as age at onset, initial white blood cell count, and National Cancer Institute risk have also no impact on OS in both cohorts. Surprisingly, the pattern of relapse in JACLS cohort, 9 of 82 patients, was unique: eight of nine patients relapsed during the maintenance phase and one patient had primary induction failure. However, bone marrow status and assessment of minimal residual disease on days 15 and 33 did not identify those patients. Interestingly, the two patients with IKZF1 deletion eventually relapsed in JACLS cohort, as did one patient in CCLSG cohort. International collaborative study of larger cohort is warranted to clarify the impact of the IKZF1 deletion on the poor outcome of TCF3-PBX1 positive BCP-ALL.

Objective: Anti-TNF-α therapy is an effective therapy for Crohn’s disease (CD). Thus anti-TNF-α therapy may alter CD64 expression, one of the Fc receptors for IgG, in CD. We investigated CD64 expression on neutrophils before and after treatment with anti-TNF-α therapy in CD. Methods: A total of 11 patients with active CD treated with anti-TNFα antibodies were enrolled. The severity of CD was assessed with the CD activity index (CDAI). Peripheral venous blood was obtained before and 2 weeks after the initial administration of anti-TNF-α antibody. CD64 expression on neutrophils was measured by FACS analysis of whole blood samples. Results: CDAI, C-reactive protein value and CD64 expression decreased significantly after anti-TNF-α therapy. Both prior to and after anti-TNF treatment, there was a significant and positive correlation between CD64 expression and CDAI or CRP. Similarly, there was a significant and negative correlation between CD64 and albumin value. Conclusion: Anti-TNF-α therapy suppresses CD64 expression of neutrophils, which may account for the mechanism underlying the efficacy of the medication in CD.

t(17;19)-acute lymphoblastic leukemia (ALL) shows extremely poor prognosis. E2A-HLF derived from t(17;19) blocks apoptosis induced by the intrinsic mitochondrial pathway and has a central role in leukemogenesis and chemoresistance. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expressed on cytotoxic T cells and natural killer cells and binds with death receptors (DR4/DR5), inducing apoptosis by dual activation of intrinsic and extrinsic pathways, and TRAIL mediates the graft-versus-leukemia (GVL) effect after allogeneic stem cell transplantation (allo-SCT). We found that cell lines and patients' samples of t(17;19)-ALL expressed death receptors for TRAIL, and recombinant soluble TRAIL immediately induced apoptosis into t(17;19)-ALL cell lines. E2A-HLF induced gene expression of DR4/DR5, which was dependent on the DNA-binding and transactivation activities of E2A-HLF through the 5' upstream region of the start site at least in the DR4 gene. Introduction of E2A-HLF into non-t(17;19)-ALL cell line upregulated DR4 and DR5 expression, and sensitized to proapoptotic activity of recombinant soluble TRAIL. Finally, a newly diagnosed t(17;19)-ALL patient underwent allo-SCT immediately after induction of first complete remission, and the patient has survived without relapse for over 3-1/2 years after allo-SCT. These findings suggest that E2A-HLF sensitizes t(17;19)-ALL to the GVL effect by upregulating death receptors for TRAIL.

Introduction Hematopoietic stem cell transplantation (HSCT) is one of the promising treatment strategies for children with refractory Langerhans cell histiocytosis (LCH), because of its immunomodulatory effects.Efficacy and indication of HSCT has been still undetermined. We analyzed the outcomes of HSCT in children with refractory LCH registered in the Transplant Registry Unified Management Program (TRUMP) conducted by the Japanese Society for Hematopoietic Cell Transplantation. Patients and methods Between 1996 and 2014, 30 patients Results Patients' characteristics The male/female ratio was 18/12. Ages of onset of LCH and at BMT were median 9 months (range 3-23 months) and median 1 years (range 0-11 years), respectively. At diagnosis of LCH, 19 patients were positive for risk organ involvement, 6 were negative. Eleven patients underwent HSCT using myeloablative conditioning (MAC) regimen, whereas 19 patients reduced intensity conditioning (RIC) regimen. Eight patients received total body irradiation (TBI) > 8Gy (10-12Gy) regimen, 3 received full dose busulfan (BU: 16mg/kg po or 17.6mg/kg iv) regimen. Seven received fludarabine (FLU: 125-180mg/m2) + melphalan (MEL: 70-180mg/m2) without TBI and 9 received FLU(120-180mg/m2)+MEL (140-180mg/m2) with low dose irradiation (2-5.1Gy) and the remaining 3 received other RIC regimens. Cyclosporine was used in 13 patients and tacrolimus was used in 16 patients for graft versus host disease (GVHD) prophylaxis. Donor sources were related donor in 9 patients and unrelated in 21 patients (cord blood 19 and bone marrow 2). Four related donors were father or mother and 3 of them were haplo-identical. In these patients, induction therapy at onset achieved a good (5/25), partial (8/25), no (4/25) and progressive (8/25) diseases. In regard to disease status at HSCT, recipients with no active diseae (NAD) were 4/25, active disease-regression (AD-r) 2/25, active disease-stable (AD-s) 4/25 and progressive (AD-p) 15/25. Eight patients received 2-chlorodeoxyadenosine (2-CdA), including 4 patients who received the combination of 2-CdA and high dose Ara-C before HSCT as salvage therapy. At HSCT, 15/23 (65%) patients were in primary induction failure and 8/23 (35%) experienced first or additional relapse, respectively. Transplantation outcomes Neutrophil recovery was observed in 24 patients and the median time to engraftment was 21 days. Platelets engraftment was observed in 17 patients and the median time to engraftment was 52 days. Acute GVHD of grade II - IV, chronic GVHD (cGVHD) were observed in 6 and 4 patients, respectively. Extensive cGVHD was observed in 3 patients who received MAC regimens (p= 0.079). Relapse after HSCT were observed 1 patient in RIC regimens and 2 patients in MAC (P=0.613). With follow-up of median 433 days (range 9-5307days) after HSCT, 17/30 (57%) patients are alive and 13 died. Death occurred within 3 months after HSCT in 8/13. The overall survival (OS) was not different between RIC and MAC (56.8% vs 63.6%, p=0.843). In regard to the correlation of disease status at HSCT and outcome, 6 patients with NAD/ AD-r had better outcome than 19 with AD-s/ AD-p (5-year OS 100% vs 52.1%, p=0.035). The 5-year FFS of the 16 patients who received FLU+ MEL based regimen were 68.8%, and were marginally better than those of the 14 patients who were conditioned with other regimens (68.8% (95%CI: 46.0-91.5) vs 42.9% (95%CI: 16.9-68.8), P=0.209). Discussion In conclusions, of 30 HSCT-recipients for refractory LCH, 17/30 (57%) are alive while post-transplant death occurred in 13/30 (43%). Our study showed that in regard to the correlation of disease status at HSCT and outcome, 6 patients with NAD/ AD-r had better outcome than 19 with AD-s/ AD-p (5-year OS 100% vs 52.1%, p=0.035). Novel bridging measures, such as targeted inhibition of the MAPK pathway, are required to stabilize the disease activity before HSCT. Disclosures No relevant conflicts of interest to declare.

免疫抑制療法に反応しない重症血球貪食性リンパ球組織球症の小児2例に持続血液透析濾過を施行した。 症例1は6歳の男児。若年性特発性関節炎の経過中に本疾患が再燃した。ステロイドパルス療法で解熱せず，低血圧，腎機能障害をきたした。 症例2は14歳の女子。EBウイルス感染による本疾患が再燃し，ステロイドパルス療法，シクロスポリン，エトポシドなどの投与でも全身状態は改善せず，低血圧，腎機能障害，播種性血管内凝固を合併した。 両症例とも持続血液透析濾過を開始後に症状と検査値が改善した。持続血液透析濾過は，低血圧，腎機能障害，播種性血管内凝固などを合併した重症血球貪食性リンパ球組織球症に対して有効であった。

Transient leukemia (TL) has been observed in approximately 10% of newborn infants with Down syndrome (DS). Although treatment with cytarabine is effective in high-risk TL cases, approximately 20% of severe patients still suffer early death. In this study, we demonstrate abundant KIT expression in all 13 patients with GATA1 mutations, although no significant difference in expression levels was observed between TL and acute myeloid leukemia. Stem cell factor (SCF) stimulated the proliferation of the TL cells from five patients and treatment with the tyrosine kinase inhibitor imatinib suppressed the proliferation effectively in vitro. To investigate the signal cascade, we established the first SCF-dependent, DS-related acute megakaryoblastic leukemia cell line, KPAM1. Withdrawal of SCF or treatment with imatinib induced apoptosis of KPAM1 cells. SCF activated the RAS/MAPK and PI3K/AKT pathways, followed by downregulation of the pro-apoptotic factor BIM and upregulation of the anti-apoptotic factor MCL1. Although we found novel missense mutations of KIT in 2 of 14 TL patients, neither mutation led to KIT activation and neither reduced the cytotoxic effects of imatinib. These results suggest the essential role of SCF/KIT signaling in the proliferation of DS-related leukemia and the possibility of therapeutic benefits of imatinib for TL patients.

JAK3 mutations have been reported in transient myeloproliferative disorder (TMD) as well as in acute megakaryoblastic leukaemia of Down syndrome (DS-AMKL). However, functional consequences of the JAK3 mutations in TMD patients remain undetermined. To further understand how JAK3 mutations are involved in the development and/or progression of leukaemia in Down syndrome, additional TMD patients and the DS-AMKL cell line MGS were screened for JAK3 mutations, and we examined whether each JAK3 mutation is an activating mutation. JAK3 mutations were not detected in 10 TMD samples that had not previously been studied. Together with our previous report we detected JAK3 mutations in one in 11 TMD patients. Furthermore, this study showed for the first time that a TMD patient-derived JAK3 mutation (JAK3(I87T)), as well as two novel JAK3 mutations (JAK3(Q501H) and JAK3(R657Q)) identified in an MGS cell line, were activating mutations. Treatment of MGS cells and Ba/F3 cells expressing the JAK3 mutants with JAK3 inhibitors significantly decreased their growth and viability. These results suggest that the JAK3 activating mutation is an early event during leukaemogenesis in Down syndrome, and they provide proof-of-principle evidence that JAK3 inhibitors would have therapeutic effects on TMD and DS-AMKL patients carrying activating JAK3 mutations.

Epstein – Barr virus (EBV) is identified as an etiological agent of African type of Burkitt’s lymphoma. Latent infection genes of EBV including EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs) are expressed in latently infected and immortalized B cells. EBV infection in immunosuppressed patients, such as human immunodeficiency virus (HIV)-infected patients or allograft recipients receiving long-term immunosuppressive therapy, leads to polyclonal B-cell proliferative disorders, frequently resulting in the development of monoclonal malignant lymphomas. These lymphomas are most often diffuse lymphomas of the B-cell type and contain EBV DNA. Salivary glands often develop chronic inflammatory lesions, sialadenitis, sometimes with the background of autoimmune conditions, and form the acquired mucosa-associated lymphoid tissue (MALT). Sialadenitis increases the risk of malignant lymphoma, and both low-grade B-cell lymphoma of MALT type and high-grade diffuse large B-cell lymphoma develop in salivary glands of sialadenitis patients [1,2]. In these cases, most of the lymphomas do not exhibit EBV genome [2]. Here, we report an infant showing the selective depletion of CD4-positive T-cells without HIV infection, who subsequently developed an EBV-positive diffuse B-cell lymphoma of left submandibular gland. A 3-month-old Japanese girl presented in December 2002 with a high fever of 388C and rubella-like skin eruptions. She was uneventful in her perinatal period. Laboratory examinations revealed a marked elevation of liver enzymes (AST, 489 IU/l; ALT 487 IU/l). The peripheral blood picture showed red

The clinical outcome of allogeneic HSCT was retrospectively analyzed in eight patients with CHS. In total, six of these eight patients are alive. Four of five patients transplanted with MAC achieved prompt engraftment, and three of the four patients, including two patients with AP before transplant, are alive without disease. In contrast, three patients transplanted with RIC without active AP disease achieved prompt engraftment and survive long term. RIC-HSCT might be an alternative treatment for CHS similar to other types of HLH, at least for patients without active AP disease.

Hypercalcemia is relatively rare but clinically important complication in childhood leukemic patients. To clarify the clinical characteristics, mechanisms of hypercalcemia, response to management for hypercalcemia, incidence of t(17;19) and final outcome of childhood acute lymphoblastic leukemia (ALL) accompanied by hypercalcemia, clinical data of 22 cases of childhood ALL accompanied by hypercalcemia (12 mg/dl) reported in Japan from 1990 to 2005 were retrospectively analyzed. Eleven patients were 10 years and older. Twenty patients had low white blood cell count (20 x 10(9)/l), 15 showed hemoglobinor =8 g/dl and 14 showed platelet countor =100 x 10(9)/l. Parathyroid hormone-related peptide (PTHrP)-mediated hypercalcemia was confirmed in 11 of the 16 patients in whom elevated-serum level or positive immunohistochemistry of PTHrP was observed. Hypercalcemia and accompanying renal insufficiency resolved quickly, particularly in patients treated with bisphosphonate. t(17;19) or add(19)(p13) was detected in five patients among 17 patients in whom karyotypic data were available, and the presence of E2A-HLF was confirmed in these five patients. All five patients with t(17;19)-ALL relapsed very early. Excluding the t(17;19)-ALL patients, the final outcome of ALL accompanied by hypercalcemia was similar to that of all childhood ALL patients, indicating that the development of hypercalcemia itself is not a poor prognostic factor.

Several approaches for the detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) have shown the importance of determining the level of MRD precisely. In the present study, we tested a new real-time quantitative polymerase chain reaction (RQ-PCR) strategy with minor groove binder (MGB) technology for immunoglobulin heavy chain gene rearrangements by positioning a MGB probe at the germline JH segments and one of the primers at the downstream introns in combination with an allele-specific oligonucleotide (ASO) primer complementary to the VH-DH or DH-JH junctional region. A MGB probe forms extremely stable duplexes with single-stranded DNA targets, allowing the use of shorter probes for hybridization-based assays. Therefore, it shows positional flexibility. We have designed two novel consensus MGB JH germline probes for analyzing all of the germline rearrangements registered in the V BASE database, and demonstrated that the MRD was detectable with the probes in 17 cases of childhood ALL. The actual copy number for the targets and dynamic changes before and after treatment were almost identical between the JH MGB probe and conventional non-MGB probes in each patient. MGB technology will undoubtedly contribute to MRD-PCR studies of childhood ALL.

This report describes a vasculitis and subsequently developing angiodestructive lymphoma in an 11-year-old Japanese-Filipino girl exhibiting mosquito allergy with the background of chronic active Epstein-Barr virus (EBV) infection. She developed necrotic skin ulcer at the site of mosquito bite, and histopathological examination revealed EBV-positive mononuclear cell infiltration throughout the wall of small-sized muscular artery. These EBV-positive lymphoid cells were oligoclonal in Southern blot analysis for EBV terminal repeats. Effectiveness of steroid therapy also supports the nonneoplastic nature. Approximately 1 year later, she developed progressive large skin ulcer without mosquito bites. Microscopically, the angiocentric or angiodestructive pattern of EBV-positive atypical cells supported the diagnosis of extranodal natural killer/T-cell lymphoma. Southern blot analysis revealed the monoclonal neoplastic nature of EBV-positive cells. In contrast to the primary mosquito bite lesion, natural killer/T-cell lymphoma cells exhibited the higher expression of EBV latent membrane protein 1 mRNA and the apparent protein expression detected by immunohistochemistry.

We developed an assay using a real-time quantitative polymerase chain reaction (RQ-PCR) for the quantitative assessment of minimal residual disease (MRD) in childhood lymphoid malignancies by using a consensus V-region probe combining a allele-specific oligonucleotide (ASO) reverse primer. Our strategy employs a set consisting of a consensus V-region probe, an ASO reverse primer, and a patient-specific forward primer for clonal antigen-receptor (IgH, immunoglobulin heavy chain; TCR, T-cell receptor) gene rearrangements (IgH-ASO and TCR-ASO RQ-PCR assays). The limit of detection in both assays was 5 copies of the target/105 cell equivalents. We tested the assays in 17 childhood malignancies (14 cases of acute lym-phoblastic leukemia and 3 of non-Hodgkin’s lymphoma). High correlation coefficients of the standard curves (>0.980) and PCR efficiency (>0.95) were achieved with all primer/probe sets. In 2 (12%) of the 17 patients, ASO primers could not be designed because there was no junctional N-sequence. The quantitative data suggest that the copy number of clonal antigen receptors markedly decreased after induction therapy in 15 of 17 patients and that 1 patient relapsed and died of the disease. Consensus probes make it possible to examine a large number of patients with only a limited number of probes. The strategy used for IgH-ASO and TCR-ASO RQ-PCR assays is accurate and reliable in the clinical prospective study of MRD in childhood lymphoid malignancies.

Incidence rates of Wilms' tumor (WT) markedly differ in East Asian and Caucasian children. In the present study, we examined WT1 deletions/mutations and loss of heterozygosity (LOH) on 11p and 11q in a large number of WTs and compared our findings with those from 4 series of Caucasian WTs. Incidence rates of the subtle WT1 mutation in 3 of the 5 series of sporadic and unilateral WTs including ours were 4.3–6.2% and similar. However, gross homozygous WT1 deletion was more frequent in our series than in some others. In addition, our series tended to show a higher incidence of LOH limited to 11p13 and a lower incidence of LOH including 11p15 than the Caucasian one. These findings indicate some genetic differences in WT between the 2 regions. One of the 4 Caucasian series reported a correlation of germinal WT1 mutation with the predominantly stromal histology. The present study not only confirms the correlation of germinal WT1 deletion/mutation with predominant stromal histology but also establishes a correlation with somatic WT1 deletion/mutations with predominant stromal histology. While WTs with WT1 abnormalities usually showed pseudodiploidy and predominant stromal histology, those without WT1 abnormalities showed various chromosome numbers and histologic subtypes. © 2001 Wiley-Liss, Inc.

Focal nodular hyperplasia of the liver is a lesion characterized by a well-circumscribed region of hyperplastic liver parenchyma and contains a stellate fibrous scar. The lesion is thought to be because of liver-cell hyperplasia that is caused by focal circulatory disturbances. We describe here a pediatric case of this lesion that provided direct histopathologic evidence of circulatory disturbances. We identified arterial and portal thrombi, as well as recanalization of arteries in the nodule. Hepatic necrosis was also seen in the lesion. We speculate that thrombosis of the hepatic artery and/or portal vein was the cause of hepatic necrosis and that reperfusion following hepatic arterial recanalization resulted in nodule formation. Although there was no stellate scar present in our case, the presence of bile ductular proliferation at the periphery of the nodule was helpful in distinguishing this lesion from adenoma and hepatocellular carcinoma. The early stage of nodular formation may explain the lack of a stellate scar in our case. The patient was treated earlier with actinomycin D and vincristine following surgical excision of Wilms' tumor. It is possible that such chemotherapy contributed to thrombosis in our case.

The chromosomal inversion (16)(p13q22), which is associated with the M4-eosinophilia subtype of human acute myeloid leukemia, causes the fusion of two distinct genes. The polypeptide encoded by the chimeric gene, PEBP2p/CBFp-SMMHC, retains the ability to interact with, and dominantly interfere with the function of proteins possessing the Runt homology domain. The Runt protein homologs constitute the DNA binding subunit of the PEBP2/CBF transcription factor. We examined the subcellular localization of PEBP2beta/CBFbeta-SMMHC, as well as that of Runt protein homologs in leukemic cells carrying inversion 16 by immunoblot analysis. A significant amount of the PEBPbeta/CBFbeta-SMMHC protein was recovered from the nuclear fraction along with the Runt protein homologs. Furthermore, some of both polypeptides was retained in the DNA pellet that represents the material remaining after extraction of nuclear fraction with high salt. These observations suggest that the so-called dominant interfering effect of PEBPbeta/CBFbeta-SMMHC on PEBP2/CBF occurs inside the nucleus. In addition, we could detect PEBP2beta/CBFbeta-SMMHC in the cytoplasmic membrane fraction as well. The function of this membrane-located PEBP2beta/CBFbeta-SMMHC, if any, appears to be unrelated to that of Runt protein homologs.

Background The primary object of this study was to identify treatment-related variables that may predict relapse of acute myelogenous leukemia (AML) after autologous peripheral blood stem cell transplantation (PBSCT), which will be critical for the development of a suitable protocol for wider application. Methods A total of 28 children (age 0-18 years) with AML underwent PBSCT and have had a minimum follow-up of 25 months; including 24 patients in their first complete remission (CR) and four in their second CR. The patients were divided into two cohorts according to the study phase: 16 patients were treated in an early phase pilot study and 12 patients in their first CR were treated in a prospective trial. Fifteen of the first-CR patients had any of the cited high-risk features of high WBC count (>100x10(9)/l; n = 5), FAB M0/M4/M5/M7 subtypes (n = 11) or delayed achievement of CR (n = 9). Except in one patient, cytoreductive regimens did not include total body irradiation (TBI). Results After PBSCT, one patient died of veno-occulsive disease (VOD) and another patient relapsed early on day 43, but the remaining patients showed engraftment. Leukemic relapse was observed 1-29 months after PBSCT (median, 8 months); in all of the 4 children treated in their second CR and in 11 of the 24 patients (46%) treated in their first CR. The remaining patients have been disease-free for 24 to 97 months (median, 53 months). Using a multivariate analysis, the timing of apheresis was the most significant prognostic factor for those treated in their first CR (p = 0.03); 12 of the 16 patients whose PBSC were collected beyond 2.5 months of CR continue to remain in CR, while seven of the eight patients whose PBSC were harvested within 2.5 months of CR relapsed. Conclusion Although the small number of patients studied does not allow firm conclusions to be drawn regarding the relative effectiveness of this therapy, the results do suggest the feasibility of further studies of PBSCT for the treatment of childhood AML with high-risk features including the assessment of minimum residual disease.

There exists limited information about the usefulness of hemopoietic stem cell transplantation (HSCT) for the treatment of patients with refractory Langerhans cell histiocytosis (LCH). We report here four Japanese pediatric patients with multisystem LCH disease who underwent HSCT between 1994 and 1997. Two of the four patients are doing well without any relapse. However, neither of them shows improved sequelae 3 to 4 years after allogeneic HSCT, although the graft was rejected in one of the cases. The remaining two patients died of septic shock. A review of the literature of 11 patients revealed four fatalities after the use of HSCT in the treatment of LCH. Three of these were due to active LCH and three deaths occurred within 2 months after HSCT. To establish the usefulness of HSCT for refractory LCH, further studies are required.

Of 40 Wilms tumors with chromosome abnormalities, 6 were hypodiploid, 10 were pseudodiploid, 7 were hyperdiploid with 47 to 49 chromosomes, and 17 were hyperdiploid with 50 or more chromosomes, mostly including +12. WT1 deletions/mutations were found in one hypodiploid, eight pseudodiploid, and one hyperdiploid (47-49 chromosomes) tumor, but in none of the hyperdiploid (> or =50 chromosomes) tumors. Of the 10 tumors with WT1 abnormalities, 6 had a homozygous WT1 deletion, 1 had a nonsense WT1 mutation and loss of heterozygosity at 11p, 1 had an intragenic hemizygous WT1 deletion without detectable WT1 mutation, and 2, which occurred in Wilms tumor-aniridia-genitourinary abnormalities-mental retardation syndrome patients, had a hemizygous deletion and a missense or frameshift mutation of WT1. Six of the nine tumors with homozygous or hemizygous WT1 deletions had chromosome aberrations involving chromosome band 11p13 in one of the two chromosomes 11. While one hypodiploid and one pseudodiploid patient died of the disease, and one hyperdiploid (47-49 chromosomes) patient was alive in nonremission, all hyperdiploid (> or =50 chromosomes) patients had no evidence of disease at the last follow-up. Our data show that chromosome aberrations are closely correlated to WT1 abnormalities and suggest that hyperdiploid (> or =50 chromosomes) Wilms tumors may be characterized by the absence of WT1 abnormalities and possibly also by a favorable prognosis.

This study reviewed the clinical characteristics of 112 pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients with TCF3-PBX1 fusion treated according to the Japan Association of Childhood Leukemia Study (JACLS) ALL02 protocol (n = 82) and Children's Cancer and Leukemia Study Group (CCLSG) ALL 2004 protocol (n = 30). The 3-year event-free survival (EFS) and overall survival (OS) rates were 85.4 ± 3.9% and 89.0 ± 3.5% in JACLS cohort, and the 5-year EFS and OS were 82.8 ± 7.0% and 86.3 ± 6.4% in CCLSG cohort, respectively, which are comparable to those reported in western countries. Conventional prognostic factors such as age at onset, initial white blood cell count, and National Cancer Institute risk have also no impact on OS in both cohorts. Surprisingly, the pattern of relapse in JACLS cohort, 9 of 82 patients, was unique: eight of nine patients relapsed during the maintenance phase and one patient had primary induction failure. However, bone marrow status and assessment of minimal residual disease on days 15 and 33 did not identify those patients. Interestingly, the two patients with IKZF1 deletion eventually relapsed in JACLS cohort, as did one patient in CCLSG cohort. International collaborative study of larger cohort is warranted to clarify the impact of the IKZF1 deletion on the poor outcome of TCF3-PBX1 positive BCP-ALL.

Children with Down syndrome have an increased incidence of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia. The majority of these cases harbor somatic mutations in the GATA1 gene, which results in the loss of full-length GATA1. Only a truncated isoform of GATA1 that lacks the N-terminal 83 amino acids (GATA1-S) remains. We found through genetic studies of 106 patients with TAM that internally deleted GATA1 proteins (GATA1-IDs) lacking amino acid residues 77-119 or 74-88 (created by splicing mutations) contributed to the genesis of TAM in 6 patients. Analyses of GATA1-deficient embryonic megakaryocytic progenitors revealed that the GATA1 function in growth restriction was disrupted in GATA1-IDs. In contrast, GATA1-S promoted megakaryocyte proliferation more profoundly than that induced by GATA1 deficiency. These results indicate that the internally deleted regions play important roles in megakaryocyte proliferation and that perturbation of this mechanism is involved in the pathogenesis of TAM.

Introduction Hematopoietic stem cell transplantation (HSCT) from an HLA-matched related donor is a first-line treatment for children with severe aplastic anemia (SAA). HSCT from an HLA-matched unrelated donor (MUD) is indicated as a salvage treatment for non-responders to immunosuppressive therapy (IST).Because unrelated cord blood transplantation (UCBT) has been considered as an experimental therapy, information of UCBT in patients with SAA is scarce, especially in pediatric patients. We analyzed the outcomes of UCBT in children with AA registered in the Transplant Registry Unified Management Program (TRUMP) conducted by the Japanese Society for Hematopoietic Cell Transplantation. Patients and Methods Between 1998 and 2013, 27 patients Results Patients' characteristics Among the 27 patients, 10 patients underwent UCBT during 1998 - 2005 and 17 patients underwent UCBT during 2006 - 2013. The median age at the time of UCBT was 6 years (range, 0 - 14). The male/female ratio was 12/15. The median interval between the diagnosis of AA and CBT was 212 days (range, 39 - 2,982). Sixteen patients received antithymocyte globulin (ATG) as immunosuppressive therapy (IST) before UCBT. The median total nucleated cell number and CD34 positive cell number at cryopreservation were 3.99 x 107/kg (range, 0.09 - 16.11 x 107/kg) and 1.31 x 105/kg (range, 0.39 - 6.17 x 105/kg), respectively. Ten patients received fludarabine (FLU) + cyclophosphamide (CY) + irradiation ± ATG. Six received FLU + CY + ATG. Four received FLU + melphalan (MEL) + irradiation and the remaining 7 received other various conditioning regimens. Cyclosporine was used in 9 patients and tacrolimus was used in 17 patients for graft-versus-host disease (GVHD) prophylaxis. Transplantation outcomes Neutrophil recovery was observed in 19 patients and the median time to engraftment was 20 days. Platelets engraftment was observed in 17 patients and the median time to engraftment was 50 days. One patient experienced secondary GF. Sustained engraftment was observed in 18 patients. Grade II - IV acute GVHD, limited chronic GVHD, and extensive chronic GVHD were observed in 6 (26%), 2 (10%), and 2 patients (10%), respectively. With median follow-up times of 18 months after CBT, 19 of 27 patients were alive. Causes of death included bacterial/fungus infections in 3, veno-occulusive disease (VOD) in 2, EBV-LPD, hematologic malignancy, and secondary GF in each 1 patient. The 5-year overall and failure-free survival (OS, FFS) of all patients were 69.5±9.0% and 59.3±9.5%, respectively. The 5-year OS and FFS of 17 patients who underwent UCBT after 2006 was 93.8±6.1% and 76.0±10.5%, respectively. Primary GF was observed in 8 patients, including 3 who underwent UCBT during 2006 - 2013. Two patients, who underwent UCBT after 2006, received FLU + CY + ATG without irradiation and remaining one patient received CY + ATG + total body irradiation (TBI). We screened anti-HLA antibodies in 8 patients but none of them were positive. The conditioning regimens including ATG exhibited a significantly worse FFS than those without ATG (33.3±12.2 % vs 91.7±8.0 %, P=0.004). On the other hand, the conditioning regimens including irradiation exhibited a better FFS than those without irradiation (75.0±9.7 % vs 14.3±13.2 % , P=0.01). The 5 year FFS of FLU + MEL + irradiation regimen (n=4) and FLU + CY + irradiation regimen (n=10) was 100% and 90%±9.5%, respectively. Univariate analysis resulted in no statistical significant differences in OS and FFS in HLA disparities, total nucleated cell number and CD34 positive cell number at cryopreservation, and methods of GVHD prophylaxis. One patient developed secondary malignancy. Discussion Our study showed that a reduced conditioning regimen with Flu, MEL/CY and low-dose irradiation may be an optimal regimen for SAA children receiving UCBT. An important finding is that ATG has no benefit in this setting. A prospective study with larger number of patients is warranted to confirm our promising results. Disclosures Kojima: SANOFI: Honoraria, Research Funding.

Since an excellent outcome was observed in a previous study (OCLSG-94) utilizing continuous cytarabine (CA) infusion in the consolidation phase for the treatment of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL), a randomization trial was performed to test the superiority of continuous CA infusion to conventional repeated CA injection as consolidation therapy in Japan Association of Childhood Leukemia Study (JACLS) ALL02 clinical trial. Newly diagnosed children with standard-risk (SR) or high-risk (HR) BCP-ALL excluding patients with Ph+ ALL and infants were included in this study. Patients showing prednisolone good response (PGR) were assigned to SR or HR based on the age at onset and initial white blood cell (WBC) counts (SR: age 1 to 9 and WBC less than 10,000 /µL, HR: remaining patients). Patients achieving complete remission (CR) at the end of induction (day 33) proceeded to the randomization. HR patients with M3 marrow on day 15 were excluded. They were randomly assigned to Arm A (cyclophosphamide 750 mg/m(2) 1hr div: day 1 and 8, CA 75 mg/m(2) 1hr div: day 1-6, 8-13 and 6MP 50 mg/m(2) orally, day 1-14) or Arm B (cyclophosphamide 500 mg/m(2) 1hr div: day 1, 3 and 5, CA 100 mg/m(2) 24hr div: day 1-5 and DEX 5 mg/m(2) 1hr div twice a day, day 1-5). Intrathecal chemotherapy (MTX, HDC and CA) was administered twice in this phase in both arms. In the HR group two doses of pirarubicin (25 mg/m (2)) were added and this phase was repeated after re-induction therapy. Estimation of event free survival (EFS) and overall survival (OS) was performed using the Kaplan-Meier method and the differences were compared using the log-rank test. Between April 2002 and May 2008, 1252 children with newly diagnosed ALL were enrolled to JACLS ALL-02 study. Among these, the number of patients with SR and HR were 388 (arm A=200, arm B=188) and 513 (arm A=251, arm B=257, not assigned=5), respectively. The characteristics of eligible patients between arm A and B were not different statistically (Table). Among SR patients, the 4y-EFS were 89.7 +/- 5.2% and 93.4 +/- 4.7% in Arm A and B, respectively (p=0.70). Among HR patients, the 4y-EFS were 89.7 +/- 5.2% and 90.5 +/- 4.4% in Arm A and B, respectively (p=0.97). Although hematological toxicity was not different, in the next phase (sanctuary phase) following Arm A consolidation therapy the frequencies of hematological grade 3 and 4 adverse effects were higher in both SR and HR (SR: p Consolidation therapy with continuous infusion of CA (Arm B) was not superior to conventional CA regimen. Disclosures No relevant conflicts of interest to declare.

Twenty percent to 30% of transient abnormal myelopoiesis (TAM) observed in newborns with Down syndrome (DS) develop myeloid leukemia of DS (ML-DS). Most cases of TAM carry somatic GATA1 mutations resulting in the exclusive expression of a truncated protein (GATA1s). However, there are no reports on the expression levels of GATA1s in TAM blasts, and the risk factors for the progression to ML-DS are unidentified. To test whether the spectrum of transcripts derived from the mutant GATA1 genes affects the expression levels, we classified the mutations according to the types of transcripts, and investigated the modalities of expression by in vitro transfection experiments using GATA1 expression constructs harboring mutations. We show here that the mutations affected the amount of mutant protein. Based on our estimates of GATA1s protein expression, the mutations were classified into GATA1s high and low groups. Phenotypic analyses of 66 TAM patients with GATA1 mutations revealed that GATA1s low mutations were significantly associated with a risk of progression to ML-DS (P < .001) and lower white blood cell counts (P = .004). Our study indicates that quantitative differences in mutant protein levels have significant effects on the phenotype of TAM and warrants further investigation in a prospective study.

We conducted a multicenter postmarketing study to investigate the efficacy and safety of reinduction therapy with a high-dose cytarabine-containing regimen for pediatric patients with relapsed or refractory acute leukemia. Seven of 13 patients (53.8%) with ALL achieved complete or partial remission, and only 1 of 6 patients (16.7%) with AML achieved partial remission. The frequent non-hematologic adverse events were gastrointestinal toxicities, such as vomiting, diarrhea and abdominal pain, as well as pyrexia and headache. Infection appeared in 9 of 20 (45%) patients. There were two death during reinduction therapy. One died of invasive bronchopulmonary aspergillosis, and the other died of intracranial hemorrhage and renal failure. These results indicated that a high-dose cytarabine regimen is effective as reinduction therapy in pediatric patients with relapsed ALL, and supportive care is essential to prevent or control treatment-related adverse events, such as infection.